POS0226 DEFICIENT CHAPERONE-MEDIATED AUTOPHAGY CONTRIBUTES TO JOINT DAMAGE IN OSTEOARTHRITIS
Authors: I. Lorenzo, U. Nogueira-Recalde, C. Garcia-Dominguez, N. Oreiro, J. A. Pinto Tasende, M. Lotz, F. J. Blanco, B. Carames
Journal: Annals of the Rheumatic Diseases (2022)

Background In Osteoarthritis (OA), defects in macroautophagy are evident and precede joint damage. Indeed, pharmacological activation of macroautophagy protects against joint damage and disease. Objectives Therefore, identifying hallmarks associated with specific autophagy subtypes could shed light to fundamental mechanisms of joint disease and facilitate the development of therapeutic strategies to prevent OA progression. Methods A gene expression analysis of 35 autophagy genes was performed from blood from a Prospective OA Cohort of A Coruña (PROCOAC) of non-OA (Age:61,44±1,16 years; BMI:25,25±0,52; Females, n=18) and knee OA subjects (Age:65,50±1,05 years; BMI:29,55±0,67; Females, n=18, OA grade III-IV) by using SYBR green array. The differential expression of candidate genes in blood (n=30/group) and knee cartilage (Non-OA, n=12; Knee OA donors, n=21) was confirmed by using Taqman Technology. HSP90AA1, a chaperone mediated autophagy (CMA) mediator, was evaluated in human knee joint tissues (i.e. cartilage, meniscus, ACL and synovium) with different KL grades (0, 2 and 4, n=3/each KL grade) and in both spontaneous aging mice (2, 6, 12, 18, and 30 months old, n=3/each time) and surgically-induced OA mice (10 weeks after surgery, n=4/each) by immunohistochemistry. The functional consequences of HSP90AA1 deficiency on inflammation, oxidative stress, senescence and apoptosis were studied in human OA chondrocytes by gene and protein expression and flow cytometry. The potential contribution of CMA to chondrocyte homeostasis was studied by assessing the capacity of CMA to restore proteostasis upon macroautophagy deficiency by ATG5 knockdown. To study the therapeutic potential of targeting CMA, HSP90AA1 was overexpressed in human OA chondrocytes. Results 16 autophagy-related genes were significantly downregulated in knee OA subjects (p&lt;0.05). Macroautophagy-related genes ATG16L2, ATG12, ATG4B and MAP1LC3B, were significantly downregulated (p&lt;0.05). Interestingly, HSP90AA1 and HSPA8, CMA mediators involved in stress response and protein folding, were significantly downregulated (p&lt;0.001). Confirmatory studies showed a downregulation of MAP1LC3B and HSP90AA1 in blood (p&lt;0.001) and cartilage (p&lt;0.05) from knee OA subjects. Moreover, HSP90A was reduced in human joint tissues (i.e. cartilage, meniscus, ACL, p&lt;0.05) and associated to OA severity. In mice, HSP90A reduction was observed not only in OA (p&lt;0.05) but also in aging (p&lt;0.01). LAMP2A, a key CMA mediator, was also reduced in human OA cartilage and associated with aging in mice (p&lt;0.05). Remarkably, HSP90AA1 deficiency was functionally linked to inflammation, oxidative stress, senescence and apoptosis (p&lt;0.05). Moreover, LAMP2A activity was decreased upon HSP90AA1 deficiency, while mTOR signaling pathway, p62 and active caspase 3 were increased (p&lt;0.05), indicating a failure in the autophagy flux that may lead to impaired lysosomal degradation and apoptosis. Human OA chondrocytes with impaired macroautophagy through ATG5 knockdown show reduced LC3II expression and induced activation of prbs6, p16 and p21 (p&lt;0.05). Interestingly, HSP90A was increased, suggesting a compensatory activation of CMA in response to specific macroautophagy defects. Remarkably, HSP90AA1 overexpression itself is sufficient to protect against joint damage by decreasing mTOR signaling and senescence in human OA chondrocytes. Conclusion Taking together, we identified HSP90A, a marker of CMA, as a key regulator of chondrocyte homeostasis underlying a relevant mechanism in OA. A better definition of the cross-talk between CMA and macroautophagy defects might reveal its role as a hallmark of OA. References [1]Caramés B, et al. Arthritis Rheum. 2010, 2015; 2. Caramés B, et al. Ann Rheum Dis. 2012 Disclosure of Interests None declared